Poster: Intracellular Signaling

Abs # 814: Calmodulin interaction with AtCNGC2 measured by FRET

Presenter:	Zielinski, Ray , zielinsk@life.uiuc.edu
Authors	Zielinski, Ray  (A)   Ditzler, Mark  (A)   Harish, Ajith  (A)
Affiliations:	(A): University of Illinois

AtCNGC2/dnd1 is a cation channel implicated in mediating hypersensitive cell death in response to pathogens. The mechanism by which it acts, however, is not understood. Calmodulin (CaM) and cyclic nucleotides bind AtCNGC2 and regulate its activity. To better understand AtCNGC2's role in the hypersensitive response we have measured CaM binding to the channel's CaM-binding domain (CaM-BD). Our goal is to decouple cyclic nucleotide and CaM regulation of the channel by altering its affinity for CaM. Fluorescent indicator proteins comprising GFP, the CNGC2 CaM-BD and BFP as well as split cameleons consisting of GFP-CNGC2 CaM-BD and BFP-CaM were used to measure CNGC2-CaM interaction. Site directed mutations in the CaM-BD were constructed and expressed that enabled us to examine the roles of strongly hydrophobic residues and regions that influence the domain's propensity for a-helix formation. We have identified mutations that increase CaM's affinity for the CNGC2 CaM-BD and those that decrease its affinity. By altering the orientations of the split cameleon interacting partners we found differences in FRET signal strength that are consistent with a "parallel" orientation in which the C-terminus of CaM binds the C-terminal domain of the CaM-BD. Current studies are aimed at introducing these mutations into the intact channel coding sequence in order to assess their effect on cyclic nucleotide regulation and channel activity. We are also introducing the fluorescent protein reporters into transgenic plants to determine whether they can be used to image CaM interaction with AtCNGC2 CaM-BD in vivo. Supported by USDA NRI (2001-353-10927).